5-Aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) fluorescence has been shown to have high tumor cell selectivity in various organs, including the gastrointestinal (GI) tract. To better understand and to possibly find new approaches to therapeutic application, we investigated the uptake kinetics and consequent metabolism of ALA and PPIX, respectively. Three colon carcinoma (CaCo2, HT29, SW480) and a stromal cell line (fibroblast, CCD18) were chosen to mimic important aspects of malignant mucosa of the GI tract. Because differential PPIX concentrations in these cell lines represented the in vivo observations (ratio tumor vs normal 10:1-20:1), we analyzed the ALA uptake, mitochondrial properties and key molecules of PPIX metabolism (porphobilinogen deaminase [PBGD], ferrochelatase [FC], iron content, transferrin receptor content). The tumor-preferential PPIX accumulation is strongly influenced, but not solely determined, by activity differences between the PPIX-producing PBGD and the PPIX-converting FC, when compared with fibroblasts. Tumor-specific PPIX accumulation is generated by ALA conversion rather than by initial ALA uptake because no significant overall difference in uptake (about 0.6 microg ALA/mg protein) of ALA is seen. In conclusion, further research of tumor cell selectivity of PPIX fluorescence should focus on the mechanisms responsible for an altered PPIX metabolism to find tumor-specific target molecules, thus leading to an improved clinical practicability of ALA application and consequent endoscopy.

5-Aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) fluorescence has been shown to have high tumor cell selectivity in various organs, including the gastrointestinal (GI) tract. To better understand and to possibly find new approaches to therapeutic application, we investigated the uptake kinetics and consequent metabolism of ALA and PPIX, respectively. Three colon carcinoma (CaCo2, HT29, SW480) and a stromal cell line (fibroblast, CCD18) were chosen to mimic important aspects of malignant mucosa of the GI tract. Because differential PPIX concentrations in these cell lines represented the in vivo observations (ratio tumor vs normal 10:1-20:1), we analyzed the ALA uptake, mitochondrial properties and key molecules of PPIX metabolism (porphobilinogen deaminase [PBGD], ferrochelatase [FC], iron content, transferrin receptor content). The tumor-preferential PPIX accumulation is strongly influenced, but not solely determined, by activity differences between the PPIX-producing PBGD and the PPIX-converting FC, when compared with fibroblasts. Tumor-specific PPIX accumulation is generated by ALA conversion rather than by initial ALA uptake because no significant overall difference in uptake (about 0.6 lg ALA/mg protein) of ALA is seen. In conclusion, further research of tumor cell selectivity of PPIX fluorescence should focus on the mechanisms responsible for an altered PPIX metabolism to find tumor-specific target molecules, thus leading to an improved clinical practicability of ALA application and consequent endoscopy.

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Photodynamic therapy using 5‐aminolevulinic acid (5‐ALA)–induced protoporphyrin IX is a promising tool in bladder‐cancer therapy. However, little is known about the cellular mechanisms of phototoxicity. Our aim was to characterize the cellular damage and to optimize differential photodynamic effectiveness between tumor and normal urothelial cells. RT4 tumor and UROtsa normal urothelial cells were used to simulate a papillary bladder tumor in contrast to normal urothelium. Photodynamically induced damage in plasma membrane and mitochondria was monitored by flow cytometry with propidium iodide exclusion and analysis of aggregate formation of the dye JC‐1. Cell morphology was investigated by phase‐contrast and fluorescence microscopy following acridine orange staining. Long incubation times (3 hr) led to complete RT4 tumor cell kill accompanied by a marked fraction of damaged normal UROtsa cells. Shorter incubation intervals (1 hr) also resulted in complete RT4 tumor cell kill; however, most UROtsa cells retained their cell properties, including intact plasma membrane and active mitochondria as well as intact cellular morphology. Phototoxicity depends not only on cellular sensitizer accumulation but also on intracellular localization. Analysis of phototoxic mechanisms is an important step for planning combination therapy regimens with, e.g., DNA‐damaging agents. Further, data indicate that differential phototoxicity in normal and tumorous urothelium can be enhanced using differences in cellular protoporphyrin IX distribution following short 5‐ALA incubation times. These data are encouraging for the in vivo situation since short incubation times are a more practical approach for local photodynamic therapy of early tumor stages not only in the bladder but also, e.g., in the gastro‐intestinal tract or bronchial mucosa. © 2001 Wiley‐Liss, Inc.